Peptide hormone is bound to the receptor of the cell membrane and transmits the information into the cells. Ca2+ plays an important role in this mechanism. Many proteins that binds Ca2+ exist in the cells, and calmodulin plays an important role as a protein that amplify that action. It is clarified that Ca2+ is bound to said calmodulin, and activates various enzymes related to the regulation of cell functions (Science, 202, 19-27, 1984). Moreover, it is known that Ca2+ acts on protein kinase C or other Ca2+ binding protein (including enzyme) (Science, 233, 305-312, 1986). Regucalcin is also a Ca2+ binding protein that was isolated from rat liver cytoplasm by the present inventor.
Regucalcin is a Ca2+ binding protein whose molecular weight is 33388, wherein the Ca2+ binding constant shows 4.19×105M−1, having 6 to 7 high-affinity Ca2+ binding sites, comprising 34% of α-helical structure, and is an acidic protein existing notably in liver, wherein the isoelectric point is pI5.20. Regucalcin is a specific protein that does not comprise a site EF hand structure (region) that is seen in calmodulin and many other Ca2+ binding proteins. For example, by binding Ca2+, the calmodulin increases its α-helical content and its structure becomes robust, but regucalcin decreases the α-helical content. Moreover, on the other hand, it has been clarified that in the regulation of cell functions, regucalcin inhibits the enzyme activation caused by calmodulin and also inhibits the activation of protein kinase C. As described, there are many knowledges such as regucalcin functions as a regulatory protein for signaling (FEBS Lett, 327, 251-255, 1993).
The regucalcin gene exists on chromosome X in rat (Xq 11.1-12), and is localized also in chromosome X in human. The regucalcin gene has been demonstrated in higher animals such as monkey, mouse, dog, bovine, rabbit, chicken and the like other than rat or human, but not yeast, and it is believed that it encodes highly differentiated protein. Regucalcin cDNA is cloned and all of its whole structure is determined (Japanese Laid-Open Patent Application No. 7-123985). As for the rat liver regucalcin cDNA, the base pair encoding whole amino acid is 0.897 kb, and it translates 299 amino acids. Furthermore, the base sequence of regucalcin cDNA of the mouse liver or human liver is also determined, and by comparison with regucalcin cDNA of rat liver, they have 94% and approximately 89% of homology, respectively. The expression of regucalcin mRNA is observed in the liver of human, rat, mouse, bovine, chicken and the like, and in these livers, the presence of regucalcin protein has been also verified.
The regucalcin is known to be a protein characterized as a regulatory protein of intracellular Ca2+ signaling having multifunctional role, and to be an important protein related to the regulation of cell functions (Life Sciences 66, 1769-1780, 2000, Biochemical and Biophysical Research Communications 276, 1-6, 2000). It has also been clarified by experiments to animals that the expression of regucalcin in liver or kidney of a living body is decreasing at the time of hepatopathy (Molecular and Cellular Biochemisty 131, 173-179, 1994) or nephropathy (Molecular and Cellular Biochemisty 151, 55-60, 1995), and the relation between regucalcin and cause of disease is suggested. There is a method to differentiate the serum of the patient having liver disease by measuring the serum of concentration of regucalcin, which exists specifically in kidney unlike the existing liver function markers such as GOT, GPT and the like, and as the regucalcin is increasing significantly in the serum of patient having liver disease, but on the other hand, almost no regucalcin is detected in the serum of a healthy person, therefore, said measurement is known to be useful for a differential diagnosis of serum of patient having liver disease (Japanese Laid-Open Patent Application No. 10-26623).
On the other hand, bone tissues are comprised of bone cells and substrates, wherein ⅓ is an organic matter wherein collagen is the major component, and ⅔ is an inorganic substance which is the bone mineral of calcium-phosphate. Structurally, it can be divided in compact substance, spongy substance and cortical substance, and for example, the diaphysis of the long bone is composed of compact substance and the epiphysis is composed of spongy substance surrounded by cortical substance. Bone is not a structure that does not change at all once after it is formed, but its structure and the amount are maintained upon the balance of bone formation and bone resorption. Therefore, in case the balance is disrupted due to aging or other reasons, various bone diseases can be developed. Among bone diseases, as for diseases developed by abnormal increase of bone resorption wherein calcium salt elutes from bone into blood vessel, malignant hypercalcemia caused by myeloma or lymphoma and the like, Paget's disease of bone caused by local bone resorption, and aosteoporosis which is not accompanied by a quality change though the absolute level of the bone is decreasing, and the like. It is known that these diseases entail bone pain and induce vulnerability of bone which can become the cause of bone fracture. These diseases are becoming serious social problem due to the increase of the population of aged person.
Furthermore, non-human mammals having DNA in which exogenous 25-hydroxyl vitamin D324-hydrozylase gene or its mutant gene is integrated, wherein the animals can be used as pathological animal model for bone disease such as hypercalcemia, hypocalcemia, hyperparathyroidism, rickets, osteomalacia, aosteoporosis, osteopenia and the like, kidney disease such as glomerulonephritis, glomerulosclerosis, chronic nephritis, renal failure and the like, malignant tumors, psoriasis or complication thereof and the like, and can be used to perform the clarification of the pathological mechanism and investigation of method of treatment of disease and screening of therapeutic agents, are known (Japanese Laid-Open Patent Application No. 11-9140).
The regucalcin protein is a specific multifunctional protein specifically expressed in liver, which is expressed also with a low level, in kidney, heart, cerebrum (neuron), is engaged with the regulation of the cell function related to intracellular Ca2+ signaling. When its expression is lowered, it induces physiological abnormality. A functional analysis has been performed heretofore by using protein or anti-regucalcin monoclonal antibody isolated from rat liver, and the present inventor has clarified functional role of regucalcin in many living body regulation, beside a role as a regulatory factor of calcium signal mentioned above, which are regulation of cell nucleus function such as regulation of calcium transportation enzyme in the cells; a role as an activating factor of protease; regulation of cell nucleus function such as: regulation of calcium transport of cell nucleus; a role in the degradation of cell nucleus DNA; a role in cell nucleus function when regenerating liver; a role in the resorption of calcium in renal tubule and the like.
The present inventor noted during the process of research for clarification of various functional roles of the regucalcin, that the regucalcin has specific effect different from many other Ca2+ binding proteins. The inventor perceived that the functional regulation of various cells to which calcium is related is formed on the balance of the expression level of regucalcin in vivo and the expression level of many other Ca2+ binding proteins such as calmodulin and the like; and had decided to examine the change and effect occurred in the living body, when the balance of the expression level of regucalcin and the expression level of many other Ca2+ binding proteins is disrupted. The object of the present invention is to provide animal model with overexpression of regucalcin, which is a tool to examine how the change and effect occur in a living body, when the balance between regucalcin and many other Ca2+ binding proteins is disrupted by overexpressing the regucalcin which is expressed primitively in the liver and the like of higher animals.
Furthermore, conventionally, concerning the calcium bone metabolism which typified by osteoporosis, as for the development of therapeutic agent and prevention of bone pathology occurring frequently in aged person especially in women, spayed rats are used. However, as for spayed animals, surgical extirpation surgery is necessary, and moreover, more than 3 months of breeding are required until the bone mass decrease. Therefore, not only the cost for research becomes expensive, but there are many technical and temporal restrictions. Furthermore, as for other animal models having bone pathology seen in clinical aspect, there are animal models having bone pathology with inflamed (rheumatism) arthritis, but as they are developed by an administration of drug, other side effects are shown and there are physiological problems. The object of the present invention is to provide animal models having bone pathology typified by osteoporosis, that can solve the above mentioned problems wherein surgical removal such as ophotectomy and the like is not necessary, that do not require breeding period until the bone mass decreases, and that do not have physiological problems such as being accompanied by side effects.
The present inventor has made a keen effort to solve the above mentioned problems. The regucalcin cDNA was cloned from rat liver cDNA library, cDNA which encodes the full length of the regucalcin protein was isolated, ORF was cut from said rat regucalcin full length cDNA to be introduced into the expression vector (pCXN2). Said gene expression vector was microinjected into the male proneucleus of the rat fertilized egg, said fertilized egg was transplanted into the uterine tube of the foster parent rat to generate rats. DNA was extracted from the tissue of the generated rats, and rats in which regucalcin cDNA is integrated were determined by PCR. From 29 rats which have been generated, 5 homogeneous rats (4 males, 1 female) expressing regucalcin cDNA were constructed, and the present inventor has found that the weight gain of said transgenic rats were significantly suppressed. Thus, the present invention has been completed.
Furthermore, the present inventor performed morphological measurement estimation of bone (bone density, bone strength, bone thickness of diaphyseal cortex, length of surrounding of cortex) and biochemical measurement estimation of bone component (amount of calcium, alkaline phosphatase activity of the marker enzyme of osteoblast, amount of DNA which is an index of the number of cells in bone tissue), with the use of bone density measurement apparatus pQTC (peripheral Quantitative Computed Tomography) for animal research, to each of the above-mentioned transformed rat that acquired an ability to overexpress regucalcin by being introduced with regucalcin gene, that do not show any disease of bone apparently. He has found that especially in femur, morphologically as well as biochemically, predominant bone pathology such as thinning of bone tissues, change of bone figuration and growth delay of coccyx due to bone resorption (dissolution of bone mineral) caused by the decreasing of bone mass and bone density are shown. He has then determined that the character of pathological model rats with overexpression of regucalcin is stable through many generations and that can bear to industrial production. Thus, he has completed the present invention.